人重组粒细胞集落刺激因子突变体rhG-CSF-Ala-17的纯化及部分理化性质

Purification and identification of recombinant human granulocyte colony stimulating factor muteint (rhG CSF Ala 17)

目的 :建立高效简便的人重组粒细胞集落刺激因子突变体rhG CSF Ala 17纯化工艺 ,并对其进行鉴定。方法 :大肠杆菌表达的rhG CSF Ala 17经包涵体洗涤、变性复性后 ,采用DEAESepharoseFastFlow阴离子交换层析和Superdex75凝胶过滤层析分离纯化。经SDS PAGE和FPLC检测纯度 ,并进行N端氨基酸序列分析、比活性分析、紫外最大吸收光谱分析、内毒素检测等指标对其进行鉴定。结果 :纯化后的rhG CSF Ala 17纯度可达99.6 4% ,比活为 1× 10 8U·mg-1。其N端 19个氨基酸分析与预期序列一致 ,紫外最大吸收光谱位于 2 80nm ,氨基酸的组成与预期结果一致 ,内毒素含量低于部颁标准。结论 :两步层析即可获得高纯度的rhG CSF Ala 17重组蛋白质 ,为进一步的rhG CSF Ala

Objective: To establish a cost effective procedure for the purification and identification of rhG CSF Ala 17. Methods: The inclusion body of the rhG CSF Ala 17 recombinant protein expressed in E.coli was washed, denatured and renatured. The two steps of chromatography (DEAE Sepharose FF ion exchange and Supexdex 75 size exclusion) were used for the purification. The purity and properties of the recombinant protein was detected by SDS PAGE, FPLC, amino acid analysis, ultravidet (UV) absorption and endotoxin analysis. Results: Following the procedure, up to 99.64% rhG CSF Ala 17 can be achieved and there is no endotoxin contamination. The bioactivity of the recombinant protein is 1×10 8U/mg; the maximal UV absorption is at 280 nm, and the N terminal amino acid analysis shows that the first 19 amino acids sequence is the same as the theoretic prediction. Conclusion: We have established a purification procedure for rhG CSF Ala 17 with highly efficiency and simplicity. It will make a base for preclinical and clinical trials of rhG CSF Ala 17.